Thursday, December 8, 2016

A different type of protein characterization -- Native + Middle-down?!?

The term "hybrid" has a lot of meanings these days. There are hybrid instruments, there are slow and lame hybrid cars (kidding, but I do give my friends a hard time over their Priuses -- especially if they used to be into tuning and/or offroading before we got all old and stuff), and there are tons of hybrid methods.

In this case, they are combining two things that -- classically were really hard to do -- intact protein analysis under Native (non-denaturing. I think there is some confusion out there as to what native mass spec is, btw) conditions PLUS doing LC-MS/MS at the "middle-down" (big pieces!) level.

The argument is this -- under denaturing LC-MS or direct injection conditions you can llose stuff (like important info or lightly associated PTMs), and you can over-complicate your spectra. What is more fun to look at, the same protein mixture that is centered around +54 charge states or the same mixture at +27?

Typically the next technique would be full out digestion with trypsin and pulling it all back together, but I think we all know how much stuff we lose there. Too many small +1 peptides and too many PTMs that will totally mess up any chances we have of getting sequence identity when trying to automatically analyze the peptides with software.

Normally when we're trying to study one protein, it is because someone wants to know EVERYTHING about it, or EVERYTHING about how it changes under their experimental conditions...

...and the Heck lab thinks that this is one way to get to those answers (and better answers!) more efficiently.